A systemic DNA sequencing strategy

A systematic DNA sequencing strategy is presented. Instead of the sequencing of randomly selected DNA fragments (the “shotgun” method), the nucleotide order is progressively determined along a DNA chain using the dideoxynucleotide termination sequencing system and the single-stranded bacteriophage vector M13 derivatives. The length of DNA along which the sequence data can be progressively read appears to be limited only by the insertion capacity of the vector. As an example of this strategy a recombinant replicative form with a 2327 nucleotide long HindIII fragment from the restriction enzyme digest of bacteriophage λ DNA was prepared. The replicative form of the recombinant was partially digested with DNAase I in the presence of Mn²⁺. As the replication origin of the phage vector was located near one end of the inserted DNA and the priming site of the vector at the other, the breaks outside the inserted DNA either destroyed the phage or removed the priming site. With the use of a unique restriction site close to the priming site, the breaks within the inserted DNA gave rise to a recombinant mixture with the inserted DNA fixed at one end and sequentially shortened at the other. Using the ddT reaction screening procedure, 11 recombinants were identified in which the inserted DNA varied in length by about 200 nucleotides. Sequencing these recombinants by the dideoxynucleotide sequencing system covered the whole 2327 nucleotides of the HindIII DNA fragment. The average number of nucleotides read from a gel was 210, which fell into the most readable region of a sequencing gel: the overlapping regions between two gels were of 33 to 48 nucleotides.     

Construction and characterization of a cDNA clone containing a portion of the bovine prolactin sequence.

Poly(A)-containing RNA from the bovine anterior pituitary has been used as a template for the enzymatic synthesis of double-stranded cDNA. The resulting double-stranded cDNA was inserted into the Pst I site of pBR322 with the oligo(dG)-oligo(dC) tailing technique and subsequently cloned in E. coli chi 1776. Clones containing sequences complementary to prolactin mRNA were identified by colony hybridization with partially purified prolactin cDNA. A 250 base pair sequence from one prolactin positive clone was extensively characterized and shown to contain the coding information for amino acids 119-192 of authentic bovine prolactin. The recombinant DNA from this clone was covalently attached to diazotized aminocellulose and used to purify prolactin mRNA from a mixture of mRNAs.     

A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously

Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation.     

B-Z DNA junctions contain few, if any, nonpaired bases at physiological superhelical densities

The reactions of bromoacetaldehyde (BAA) with recombinant plasmids that contain sequences which can adopt left-handed Z structures or, at other locations, cruciforms were studied as a function of supercoil density. The sequence in pRW756 that undergoes a supercoil induced transition from a right to left-handed helix was (dC-dG)16 and regions near the replication origin of the pBR322 vector were converted from linearforms to cruciforms. The locations of the most nonpaired structural features were mapped by S1 nuclease cleavage of the "wedged open" duplexes after linearization of the DNAs. Three cruciforms in the pBR322 portions of the plasmids were specifically detected by BAA reaction at physiological supercoil densities (sigma = -0.067). However, the B-Z junctions did not react with BAA under these conditions although the junctions were present since the (dC-dG)16 was shown to be left-handed. Thus, the B-Z junctions have less single-stranded character than the pBR322 cruciforms (3-6 nonpaired bases) and may be fully paired. At much higher superhelical densities (sigma = -0.11-0.12), the B-Z junctions as well as the cruciforms react with BAA indicating a change in the nature of the junctions. Studies were also performed with pRW777 which harbors the mouse kappa immunoglobin sequence (dT-dG)32 . (dC-dA)32 that adopts a left-handed helix under appropriate conditions; the results were similar to those found with pRW756.     

Search for the optimal sequence of the ribosome binding site by random oligonucleotide-directed mutagenesis.

Synthetic DNA duplexes corresponding to the ribosome binding site (RBS) were synthesized through the phosphite method on solid support. The synthetic RBS DNA with partial random sequences was inserted into an appropriate site between the lpp-lac promoter and the beta-galactosidase structural gene in plasmid pMKT2. The level of beta-galactosidase expression was correlated with the color intensity of the recombinant colonies on X-gal plates. The bluest colonies were isolated and characterized with respect to beta-galactosidase enzyme activity and RBS sequence. There was good correlation between color intensity and the level of the enzyme activity, and this provided a reliable phenotypic screening method in the search for the optimal regulatory sequences. Novel RBS sequences obtained here show not only the unique nucleotide distribution, but also strong complemetarity to the 3' end region of 16S rRNA, from which could be deduced a generalized RBS sequence, the position of the SD region, and the 16S rRNA position mediated during translation initiation.     

Genetic instability of an artificial palindrom DNA sequence

A short DNA palindrom, produced by head to head ligation of a 29 bp DNA fragment, was inserted into a 27,000 bp plasmid DNA element composed of two functional replicons (R6K, ColE1). Several plasmid types containing a single copy of this palindrom in different locations of insertion on the R6K sequence were obtained. The palindrom was engineered to possess a unique EcoRI recognition sequence at its axis of symmetry. The presence of this restriction site allowed to monitor the genetic stability of the artificial palindrom at their different insertion loci. Out of 5 different insertion locations, one (in pAS807) was found to lead to a significant destabilization of the palindrom. This insertion site lies within the replication control region of R6K. We have shown that the inserted palindrom in pAS8O7 does not affect the functionality of the R6K replication origins. Excission of the palindrom sequences from pAS8O7 was not accompanied by loss of the adjacent R6K DNA sequences. Different deletion derivatives of pAS807 were generated in-vitro in order to determine the driving unit of DNA sequences around the palindrom that are involved in its excision. The results imply that large DNA structure(s) around the palindrom are involved in its excission. Complete deletion of R6K sequences from either the left or the right side of the palindrom resulted in new configurations which stabilized the palindrom. A configuration of R6K DNA sequences exceeding 270 bp long sequence from both sides of the palindrom are necessary for the transition from a palindrom stable to palindrom unstable state. In addition evidence is presented to show that the excision process of palindrom sequences requires a functional polymerase I but not the gene product of recA.     

Synthesis of DNA coding for human proinsulin

A chemical-enzymatic synthesis of 271- and 286-bp DNA duplexes, each of which contains the entire sequence coding for human proinsulin has been accomplished. In addition to the coding sequence, the 271-bp fragment carries translation initiation and termination signals plus EcoRI-HindIII restriction enzyme sites for insertion into an appropriate plasmid vector. The 286-bp fragment also contains a Shine-Dalgarno (SD) sequence preceding an ATG codon. Employing the 286-bp polynucleotide, the 568-bp tandem proinsulin gene has been obtained. The synthesis of these DNA fragments involved preparation of 42 oligonucleotides by a rapid N-methylimidazolide phosphotriester method and enzymatic conversion of the oligonucleotides into the gene subfragments, which were cloned separately and fused to yield the desired DNAs coding for proinsulin. The proinsulin gene fragments were cloned in Escherichia coli and shown to have the correct sequences.     

Bleomycin-A2 complexes with poly(dA--dT): a proton nuclear magnetic resonance study of the nonexchangeable hydrogens

The mRNA coding for uteroglobin, a progesterone-induced uterine protein, has been partially purified from 4-day pregnant rabbit uterus. Double-stranded DNA synthesized from the partially purified mRNA preparation was inserted into the Pst I site of pBR 322. Bacterial transformants containing uteroglobin DNA sequences were identified by their ability to enrich for uteroglobin mRNA on hybridization with total uterine poly A-RNA. The identity of one recombinant was confirmed unambiguously by matching its nucleotide sequence with the amino acid sequence of the uteroglobin polypeptide.     

Specificity from the synapsis of DNA elements by the Sfi I endonuclease.

The synapsis of DNA sites is a prerequisite for the reactions of many proteins that act at specific DNA sequences. The requirement for synapsis was investigated by analysing the reactions of Sfi I, a tetrameric restriction enzyme that cleaves DNA only after interacting with two recognition sites. In the presence of Mg2+, oligonucleotide duplexes with the cognate recognition sequence were cleaved rapidly, with cooperative kinetics, while non-cognate duplexes were not cleaved. In the absence of Mg2+, the primary complex formed by Sfi I with cognate DNA contained two duplexes synapsed by the tetramer: a secondary complex containing one duplex was seen only at elevated Sfi I concentrations. In contrast, the principal complex with non-cognate DNA contained one duplex bound to Sfi I. Pairs of non-cognate duplexes, or one cognate and one non-cognate duplex, generally failed to form synaptic complexes. On adding Mg2+to complexes with cognate DNA, cleavage occurred much more rapidly in the synaptic complex than in the secondary complex. DNA synapsis thus acts to enhance the specificity of Sfi I for its recognition sequence, by demanding two cognate sites for a catalytically active complex and by excluding non-cognate sites from the synaptic complex.     

The integrated forms of the S1 and S2 DNA elements of maize male sterile mitochondrial DNA are flanked by a large repeated sequence.

The mitochondrial DNA of maize was cloned using the cosmid, Homer I. Recombinants carrying sequences homologous to the S1 and S2 DNA elements of male sterile maize have been analysed. Restriction endonuclease maps for Sac II, Sma I and Bam HI have been constructed. The S1 and S2 sequences are single copy sequences occurring at unique sites; each is flanked by a 26 kb repeated sequence. The repeated sequence has been shown not to contain the mitochondrial ribosomal RNA genes.     

Construction and analysis of recombinant DNAs containing a structural gene for rat prolactin.

Poly A-containing RNA enriched in prolactin-coding sequences was isolated from female rate pituitaries after induction with diethylstilbesterol. Double stranded cDNA was synthesized from this RNA and inserted into plasmid pBR322 at the Pst I site via the poly(dG):polyy(dC) tailing method. E. coli was transformed with this DNA and the recombinant plasmid in one of the transformants characterized in detail. About half of its 900 base pair cDNA insert was sequenced. The DNA sequence is consistent with most of the reported amino acid sequence of rat preprolactin. In addition, the recombinant plasmids in two of the other transformants appear to contain growth hormone coding sequences.     

Effective method of oligonucleotide-controlled mutagenesis of DNA fragments

A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency. The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2. These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites. The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites. The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends. This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase). The digestion under controlled conditions produces duplex with protruding single-stranded 5'-regions which include the site of the desired mutation. The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex. The closed circular heteroduplex is formed by ligation. After transformation into E. coli, DNA replication generates homoduplexes, some of which contain the mutation. Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants. The yield of the mutants is 10-20%. This technique can be extended to replicative form of M13 vectors. It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends.     

Genetic Instability of an Artificial Palindrom DNA Sequence

Abstract

A short DNA palindrom, produced by head to head ligation of a 29 bp DNA fragment, was inserted into a 27,000 bp plasmid DNA element composed of two functional replicons (R6K, ColE1). Several plasmid types containing a single copy of this palindrom in different locations of insertion on the R6K sequence were obtained.(l) The palindrom was engineered to possess a unique EcoRI recognition sequence at its axis of symmetry. The presence of this restriction site allowed to monitor the genetic stability of the artificial palindrom at their different insertion loci. Out of 5 different insertion locations, one (in pAS807) was found to lead to a significant destabilization of the palindrom. This insertion site lies within the replication control region of R6K. We have shown that the inserted palindrom in pAS807 does not affect the functionality of the R6K replication origins. Excission of the palindrom sequences from pAS807 was not accompanied by loss of the adjacent R6K DNA sequences. Different deletion derivatives of pAS807 were generated in-vitro in order to determine the driving unit of DNA sequences around the palindrom that are involved in its excision. The results imply that large DNA structure(s) around the palindrom are involved in its excission. Complete deletion of R6K sequences from either the left or the right side of the palindrom resulted in new configurations which stabilized the palindrom. A configuration of R6K DNA sequences exceeding 270 bp long sequence from both sides of the palindrom are necessary for the transition from a palindrom stable to palindrom unstable state. In addition evidence is presented to show that the excision process of palindrom sequences requires a functional polymerase I but not the gene product of recA.     

Base pairing involving deoxyinosine: implications for probe design.

The thermal stability of oligodeoxyribonucleotide duplexes containing deoxyinosine (I) residues matched with each of the four normal DNA bases were determined by optical melting techniques. The duplexes containing at least one I were obtained by mixing equimolar amounts of an oligonucleotide of sequence dCA3XA3G with one of sequence dCT3YT3G where X and Y were A, C, G, T, or I. Comparison of optical melting curves yielded relative stabilities for the I-containing standard base pairs in an otherwise identical base-pair sequence. I:C pairs were found to be less stable than A:T pairs in these duplexes. Large neighboring-base effects upon stability were observed. For example, when (X,Y) = (I,A), the duplex is eight-fold more stable than when (X,Y) = (A,I). Independent of sequence effects the order of stabilities is: I:C greater than I:A greater than I:T congruent to I:G. This order differs from that of deoxyguanosine which pairs less strongly with dA; otherwise each deoxyinosine base pair is less stable than its deoxyguanosine counterpart in the same sequence environment. Implications of these results for design of DNA oligonucleotide probes are discussed.     

The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in vivo

The nature of specific DNA sequences that arrest synthesis by mammalian DNA polymerase alpha in vitro was analyzed using circular, single-stranded M13 or phi X174 virion DNA templates annealed to a unique, terminally labeled, DNA primer. This method rigorously defined both the starting nucleotide position and the direction of synthesis, as well as making the amount of radioactivity proportional to the number rather than the length of nascent DNA chains. The precise nucleotide locations of arrest sites were determined over templates with complementary sequences by cloning unique DNA restriction fragments into M13 DNA and isolating virions containing either the Watson or Crick strand. Results were correlated with the locations of palindromic (self-complementary) sequences, repeated sequences, and repeated sequences with mirror-image orientation. Two classes of DNA synthesis arrest sites were identified, distinct in structure but equivalent in activity. Class I sites consisted of palindromic sequences that formed a stable hairpin structure in solution and arrested DNA polymerase on both complementary templates. The polymerase stopped precisely at the base of the duplex DNA stem, regardless of the direction from which the enzyme approached. Class II sites consisted of non-palindromic sequences that could not be explained by either secondary structure or sequence symmetry elements, and whose complementary sequence was not an arrest site. Size limits, orientation and some sequence specificity for arrest sites were suggested by the data. Arrest sites were also observed in vivo by mapping the locations of 3'-end-labeled nascent simian virus 40 DNA strands throughout the genome. Arrest sites closest to the region where termination of replication occurs were most pronounced, and the locations of 80% of the most prominent sites appeared to be recognized by alpha-polymerase on the same template in vitro. However, class I sites were not identified in vivo, suggesting that palindromic sequences do not form hairpin structures at replication forks.     

Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes.

To improve the previous DNA/DNA nearest-neighbor parameters, thermodynamic parameters (deltaH degrees, deltaS degrees and deltaG degrees) of 50 DNA/DNA duplexes were measured. Enthalpy change of a helix initiation factor is also considered though the parameters reported recently did not contain the factor. A helix initiation factor for DNA/DNA duplex determined here was the same as that of RNA/RNA duplex (deltaG degrees(37) = 3.4 kcal/mol). The improved nearest-neighbor parameters reproduced not only these 50 experimental values used here but also 15 other experimental values obtained in different studies. Comparing deltaG degrees(37) values of DNA/DNA nearest-neighbor parameters obtained here with those of RNA/RNA and RNA/DNA, RNA/RNA duplex was generally the most stable of the three kinds of duplexes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA duplexes is sequence dependent.